The long-term goal of this project is to understand the mechanisms by which neurons control the Ca currents in their surface membranes. The Ca currents are directly responsible for the intracellular Ca signals that trigger neurotransmitter release, modulate membrane excitability and control neurite growth. This project uses molluscan neurons, which provide excellent models for studying transmitter release (e.g. squid giant synapse) and the cellular basis of behavior (e.g. Aplysia learning). The large molluscan neurons allow the application of a broad range of biophysical techniques; accurate measurement of membrane currents is possible either when intracellular environment is controlled (internal perfusion and patch clamp techniques) or minimally disturbed (two-electrode voltage clamp). All studies proposed for this period use isolated neurons from the snail Lymnaea stagnalis. The intensity and time course of the intracellular Ca2+ signal depends on two properties of the Ca channels, their activity and their distribution. The first part of this project focuses on intracellular control of the activity of Ca channels. Molluscan Ca currents are blocked by intracellular Ca2+ (Ca-dependent inactivation) and are very liable when exposed to an artificial intracellular solution (washout). The hypothesis that block by intracellular Ca2+ and washout of Ca currents is mediated by dephosphorylation of the channel will be tested. The inactivation of Ca current in patches will be studied to determine if Ca channels have to be clustered to exhibit Ca-dependent inactivation. Photorelease of Ca2+ will be used to measure both the concentration dependence and time course of block of Ca current by intracellular Ca2+. The second part of the project focuses on the distribution of Ca channels in the neuronal membrane and the relation of Ca-activated channels to the Ca channels. The microscopic distribution of Ca channels will be determined by simultaneous measurements of patch capacitance and patch Ca current. Fura-2 imaging will be used to measure spatial gradients of Ca2+ during internal perfusion of neurons and the apparent redistribution of Ca channels that occurs in cultured cells. The relative location of Ca- activated K channels will be studied, and the role of Ca-activated divalent permeable channel in controlling intracellular Ca2+ will be clarified.